A confocal laser microscope is configured such that laser light is focused on a specimen through an objective lens, a light flux of reflected light, scattered light, or fluorescent light generated on the specimen is transmitted by an optical system, and the light flux transmitted through a pinhole disposed at an optically conjugated position with respect to a light focusing point on the specimen is received on a detector. Disposing the pinhole makes it possible to filter the light generated on the specimen other than the light focusing point. Therefore, the confocal laser microscope can acquire an image with a good S/N ratio.
Further, the confocal laser microscope is configured to acquire a planar image of a specimen by scanning the specimen with laser light along two directions (X-direction and Y-direction) orthogonal to each other, along a plane perpendicular to the optical axis. On the other hand, the confocal laser microscope is configured to acquire a plurality of tomographic images (Z-stack images) along the Z-direction by changing the distance along the optical axis direction (Z-direction) between the objective lens and the specimen, whereby a three-dimensional image of the specimen is formed.
In observing a biospecimen, it is often the case that the biospecimen is observed through a cover glass in a state in which the biospecimen is immersed in a broth. Further, generally, the objective lens is designed so that an optimum imaging performance at a position immediately below the cover glass is best. In observing the inside of a biospecimen, it is preferable to acquire an image transmitted through a broth or biological tissues and having a certain depth at an observation position. Aberrations are generated in proportion to the distance from the position immediately below the cover glass to the observation position, and as a result, the resolution may be lowered.
Further, the cover glasses have variations in the thickness thereof within the tolerance range from the design value (e.g. 0.17 mm). Aberrations are generated in proportion to a difference between the actual thickness of the cover glass and the design thickness due to a difference between the refractive index (=1.525) of the cover glass and the refractive index (=1.38 to 1.39) of the biospecimen. Further, when the objective lens is an immersion lens, aberrations are generated in proportion to the depth of a biospecimen with respect to the observation position due to a difference between the refractive index of the biospecimen and the refractive index (=1.333) of water in the same manner as described above. As a result, the resolution to be obtained in observing a deep part of the biospecimen may be lowered.
As one way of solving the above defects, a correction ring has been proposed (e.g., see Patent literature 1). The correction ring is a ring-shaped rotary member provided for an objective lens, and distances between lens groups constituting the objective lens are changed by rotating the correction ring. Aberrations due to an error in the thickness of the cover glass or observing a deep part of the biospecimen are cancelled by rotating the correction ring. A scale is marked on the correction ring. For instance, rough numerical values such as 0, 0.17, and 0.23 are indicated concerning the thickness of the cover glass. Then, adjusting the scale of the correction ring in accordance with a thickness of an actually used cover glass makes it possible to adjust the distances between the lens groups in such a manner as to optimize the distances in accordance with the thickness of the cover glass.
However, the operation of the correction ring is performed by manually rotating a ring-shaped adjustment mechanism provided on the objective lens. Therefore, focus deviation or view field deviation resulting from adjusting the adjustment mechanism may occur. Further, to determine an optimum position of the objective lens, it is preferable to repeat the operation of the correction ring and focusing, resulting in a process for the optimization being cumbersome. Since the process is cumbersome, it takes time to make adjustments in order to obtain an optimum position, and a fluorescent pigment may fade. The fading of the fluorescent pigment is a problem of weakening fluorescent intensity due to continuous emission of excitation light.
Further, the operation of the correction ring may need fine control. Under the present circumstances, judgment on the adjustment result of the focusing by the operation relies on a person who visually observes an image and therefore, it is very difficult to judge whether the objective lens is located at an optimum position. In particular, in photographing images of Z-stack, it is preferable to repeat this operation for the number of images acquired in a depth direction, which is very cumbersome. As a result, under the present circumstances, the number of users who sufficiently utilize the correction ring is small. Further, in some specimens, vibrations resulting from touching may affect the observation position, and in view of the above, it is desirable to automatically adjust the focus without touching the objective lens by hand.
A technique is proposed in which, without the need of touching an objective lens or its frame by hand, a user corrects wave front aberrations generated by an optical system depending on the specimen or observation conditions, by using a phase modulation device which is disposed in the optical system including the objective lens and which displays a phase modulation profile having a polarity opposite to the polarity of a phase distribution, which profile is represented according to a relational equation between a numerical aperture of the objective lens and the ratio of the third-order spherical aberration and the fifth-order spherical aberration given when the phase distribution of the wave front aberrations is resolved using Zernike polynomials (e.g., see Patent literature 2).